Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames test: non mutagenic with and without metabolic activation (OECD 471, GLP, K, rel. 1)

- Micronucleus test in human lymphocytes: non clastogenic with and without metabolic activation (OECD 487, GLP, K, rel. 1 and read-across with Lavdender oil, equivalent to OECD 487, K rel.2).

- HPRT test in L5178Y cells: non mutagenic with and without metabolic activation (OECD 476, GLP, K, rel. 1).

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 22 September to 11 October, 2019.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted in compliance with OECD Guideline No. 471.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997.
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
Inspected on 2018-06-19,19&20 / Signed on 2018-09-21.
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : Molecular Toxicology Incorporated, Boone, NC, USA (MolTox™). The batch of S-9 used in this study was 3945, which had a protein content of 37 mg/mL.
- method of preparation of S9 mix : according to the method of Ames et al from male Sprague Dawley rats induced with a single dose of Aroclor 1254.
- concentration of S9 in the final culture medium: S9-fraction 10% v/v
- quality controls of S9: enzymatic activity, sterility, metabolic capability (certificate included in the study report)
- other: The S-9 mix was prepared immediately before use and kept on ice.
Test concentrations with justification for top dose:
Cytotoxicity test:1.5, 5, 15, 50, 150, 500, 1500 or 5000 μg/plate in TA98 and WP2 uvrA in the presence and absence of S-9 mix, under the direct plate incorporation method.

Mutagenicity test:
Main test: the doses of Essential Oil of Lavandin Grosso used in Experiment 1, under plate incorporation conditions, were as follows:
• 1.5, 5, 15, 50, 150 or 500 μg/plate in TA1537 in the absence of S-9 mix
• 5, 15, 50, 150, 500 or 1500 μg/plate in TA1535, TA98 and TA100 in the absence of S-9 mix and all Salmonella strains in the presence of S-9 mix
• 15, 50, 150, 500, 1500 or 3000 μg/plate in WP2 uvrA in the absence of S-9 mix
• 15, 50, 150, 500, 1500 or 5000 μg/plate in WP2 uvrA in the presence of S-9 mix

Confirmation test: the doses of Essential Oil of Lavandin Grosso used in Experiment 2, under pre-incubation conditions, were as follows:
• 0.5, 1.5, 5, 15, 50 or 150 μg/plate for TA1537 and TA100 in the absence of S-9 mix
• 1.5, 5, 15, 50, 150 or 500 μg/plate for TA1535 and TA98 in the absence of S-9 mix and TA1535, TA1537 and TA100 in the presence of S-9 mix
• 5, 15, 50, 150, 500 or 1500 μg/plate for WP2 uvrA in the absence of S-9 mix and TA98 in the presence of S-9 mix
• 15, 50, 150, 500, 1500 and 5000 μg/plate for WP2 uvrA in the presence of S-9 mix
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: no data. The test item was formulated on the day of use as a solution in dimethylsulphoxide (DMSO), at concentrations up to 50 mg/mL.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
Remarks:
without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-amino-anthracene
Remarks:
with S9 mix
Details on test system and experimental conditions:
SOURCE OF TESTER STRAINS: Strains of salmonella typhimurium and E. Coli were obtained from Molecular Toxicology Incorporated, Boone, NC, USA (MolTox™).

METHOD OF APPLICATION: in agar (plate incorporation) and preincubation

DURATION
- Preincubation period: 20 min at 37+/-3 °C
- Exposure duration: 37+/-3 °C for 66 hours

NUMBER OF REPLICATIONS: 3 plates/dose (mutation tests) and 2 plates/dose (range-finder).

DETERMINATION OF CYTOTOXICITY
- Method: A reduction in the number of colonies in a dose-dependent manner compared to negative control for any strain and condition might indicate cytotoxicity.

OTHER:
- After an incubation of about 66 hours at about 37 ºC, the number of colonies per plate was counted.
The number of revertant colonies per plate was counted and recorded by an automatic colony counter. Average plate counts was presented with the mean and the standard deviation for each set of triplicates or diplicates (range-finder) per test item concentration and was used to calculate the ratio of colonies per exposed plate compared to the corresponding negative control.
- Test item solubility: soluble in ethanol
Evaluation criteria:
For the test to be considered positive, the test item would have had to induce a concentration related increase in mean revertants of at least twice the concurrentvehicle control for TA98, TA100 or WP2 uvrA or at least three times the concurrent vehicle control for TA1535 or TA1537. A clear positive response in a single experiment would be sufficient to determine the result, particularly if multiple strains were affected.
As the results met the criteria for a clear negative response, statistical test results were not taken into consideration.
Statistics:
Mean, standard deviation (s.d.), fold increases compared with negative Controls and Dunnett's t-statistic (t) were calculated for each group and bacterial strain.
Key result
Species / strain:
bacteria, other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Test item was found to be soluble when diluted in ethanol.
- Precipitation: None
- Other confounding effects: None

CYTOTOXICITY TEST:
- Range finder: the doses selected for the mutation experiments were based on the toxicity of the test item or were chosen to include the guideline regulatory maximum dose level (5000 μg/plate)
* Cytotoxicity (Experiment 1):
Essential Oil of Lavandin Grosso was analysed up to the limit of toxicity of 150 μg/plate in TA1537 in the absence of S-9 mix, 500 μg/plate in TA1535, TA98 and TA100 in the absence of S-9 mix and all Salmonella strains in the presence of S-9 mix, and 1500 μg/plate in WP2 uvrA in both the presence and absence of S-9 mix, under plate incorporation conditions.
There was a reduction (to less than 0.5 times the negative Control value) in the mean colony count in TA100 at 500 μg/plate in the presence of S-9 mix, indicating toxicity of the test item to the bacteria.
* Cytotoxicity (Experiment 2):
Essential Oil of Lavandin Grosso was analysed up to the limit of toxicity of 150 μg/plate in TA1535, TA1537 and TA100 in the absence of S-9 mix, 500 μg/plate in TA98 in the absence of S-9 mix and all Salmonella strains in the presence of S-9 mix, and 1500 μg/plate in WP2 uvrA in both the presence and absence of S-9 mix, under pre-incubation conditions.
There was a reduction (to less than 0.5 times the negative Control value) in the mean colony count in WP2 uvrA at 1500 μg/plate in the presence of S-9 mix, indicating toxicity of the test item to the bacteria.

MUTAGENICITY TEST:
- There were no increases in revertant numbers, greater than the defined fold-increases (twice the negative Control value for TA98, TA100 or WP2 uvrA or three times the negative Control value for TA1535 or TA1537), compared with the relevant negative Control values in any strain at any dose level of the test substance, in the presence or absence of S-9 mix.

HISTORICAL CONTROL DATA
- Positive and negative controls showed absolute numbers of revertant colonies comparable to historical data of the test facility.



Conclusions:
Under the test condition, test substance is not mutagenic with and without metabolic activation in S. typhimurium strains TA1535, TA1537, TA98 and TA100, and E.coli WP2uvrA.
Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and Escherichia coliWP2(pKM101) were exposed to the test substance diluted in dimethylsulphoxide (DMSO) at the following concentrations both in the presence and absence of metabolic activation system (10% v/v S9).

Cytotoxicity test: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate in TA98 and WP2 uvrA in the presence and absence of S-9 mix under the direct plate incorporation method. 

Mutagenicity test:

Experiment 1 (direct plate incorporation method):

- 1.5, 5, 15, 50, 150 or 500 μg/plate in TA1537 in the absence of S-9 mix

- 5, 15, 50, 150, 500 or 1500 μg/plate in TA1535, TA98 and TA100 in the absence of S-9 mix and all Salmonella strains in the presence of S-9 mix

- 15, 50, 150, 500, 1500 or 3000 μg/plate in WP2 uvrA in the absence of S-9 mix

- 15, 50, 150, 500, 1500 or 5000 μg/plate in WP2 uvrA in the presence of S-9 mix

Experiment 2 ( pre-incubation method):

- 0.5, 1.5, 5, 15, 50 or 150 μg/plate for TA1537 and TA100 in the absence of S-9 mix

- 1.5, 5, 15, 50, 150 or 500 μg/plate for TA1535 and TA98 in the absence of S-9 mix and TA1535, TA1537 and TA100 in the presence of S-9 mix

- 5, 15, 50, 150, 500 or 1500 μg/plate for WP2 uvrA in the absence of S-9 mix and TA98 in the presence of S-9 mix

- 15, 50, 150, 500, 1500 and 5000 μg/plate for WP2 uvrA in the presence of S -9 mix.

Vehicle and positive control groups were also included in mutagenicity tests.

Positive and negative controls showed absolute numbers of revertant colonies comparable to historical data.  

There was a marked reduction in the mean colony count in TA100 at 500 μg/plate in the presence of S-9 mix in Experiment 1 and in WP2 uvrA at 1500 μg/plate in the presence of S-9 mix in Experiment 2, indicating toxicity of the test item to the bacteria. No precipitation was evident in either experiment.

There were no increases in revertant numbers, greater than the defined fold-increases, compared with the relevant negative Control values in any strain at any dose level of Essential Oil of Lavandin Grosso, in the presence or absence of S-9 mix, under either plate incorporation

or pre-incubation conditions.

 

Under the test condition, test substance is not mutagenic with and without metabolic activation inS. typhimuriumstrains TA1535, TA1537, TA98 and TA100, and E.coli WP2uvrA.

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 March to 24 May 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD Guideline 476 without any deviation.
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of assay:
other: in vitro gene mutation study in mammalian cells
Target gene:
hprt locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Dr Donald Clive, Burroughs Wellcome Co.

MEDIA USED
- Type and identity of media including CO2 concentration:
RPMI A: Horse serum (heat inactivated, 0 % v/v). Penicillin (100 units/mL), streptomycin (100 μg/mL), amphotericin B (2.5 μg/mL), Sodium pyruvate acid (0.2 mg/mL) and pluronic (0.5 mg/mL)
RPMI 10: Horse serum (heat inactivated, 10 % v/v), penicillin (100 units/mL), streptomycin (100 μg/mL), amphotericin B (2.5 μg/mL), Sodium pyruvate acid (0.2 mg/mL) and pluronic (0.5 mg/mL)
RPMI 20: Horse serum (heat inactivated, 20 % v/v), penicillin (100 units/mL), streptomycin (100 μg/mL), amphotericin B (2.5 μg/mL) and Sodium pyruvate acid (0.2 mg/mL)
RPMI 5 consisted of RPMI 10 diluted with RPMI A [prepared as RPMI 10 but with no serum added] to give a final concentration of 5% serum

CELL CULTURES
- For each experiment, at least one vial was thawed rapidly, the cells diluted in RPMI 10 and incubated at 37 ± 1 °C. When the cells were growing well, subcultures were established in an appropriate number of flasks.
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically 'cleansed' against high spontaneous background: Yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
2 % S9 (final concentration); S9 fraction was prepared from liver homogenates of male Sprague Dawley rats treated with Aroclor 1254.
Test concentrations with justification for top dose:
Range-Finder Experiment: 39.06, 78.13, 156.3, 312.5, 625 and 1250 μg/mL, with and without S9 mix
Justification: A maximum concentration of 1250 μg/mL was therefore selected for the cytotoxicity Range-Finder Experiment in order that treatments were performed up to a precipitating treatment concentration (OECD, 2016).

Mutation Experiment:
Without S9: 40, 60, 80, 100, 120, 130, 140, 150, 160, 170, 180 and 200 μg/mL
With S9: 50, 100, 150, 200, 220, 240, 260, 280, 300, 350, 400 and 500 μg/mL
Justification: Concentrations selected for the Mutation Experiment were based on the results of this cytotoxicity Range-Finder Experiment.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: Preliminary solubility data indicated that test item was soluble in anhydrous analytical grade DMSO at concentrations up to at least 507 mg/mL. The solubility limit in culture medium was in the range of 158 to 317 μg/mL, as indicated by precipitation at the higher concentration which persisted for at least 3 hours after test item addition.
- Test substance preparation: Test item stock solutions were prepared by formulating test item under subdued lighting in DMSO, with the aid of vortex mixing, as required, to give the maximum required concentration. Subsequent dilutions were made using DMSO. The test item solutions were protected from light and used within approximately 2 hours of initial formulation.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO diluted 100-fold in the treatment medium
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO diluted 100-fold in the treatment medium
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; RPMI 1640 media supplied containing L-glutamine and HEPES

- Cell density at seeding:
Cytotoxicity Range-Finder Experiment: Cell concentrations were adjusted to 8 cells/mL and, for each concentration, 0.2 mL was plated into each well of a 96-well microtitre plate for determination of relative survival.
Mutation Experiment: At least 10^7 cells in a volume of 18.8 mL of RPMI 5 were placed in a series of sterile disposable 50 mL centrifuge tubes.

DURATION
- Exposure duration: 3 h
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 14 days
- Plating for Survival: 7 days
- Plating for viability: 8 to 9 days
- All incubations were performed at 37 ± 1 °C in a humidified incubator gassed with 5 ± 1 % v/v CO2 in air.

SELECTION AGENT (mutation assays): 6-thioguanine (6TG) at a final concentration of 15 μg/mL

NUMBER OF REPLICATIONS:
- Preliminary toxicity test: Single cultures/dose for test item and vehicle control
- Main test: Two cultures for vehicle control and test item; single culture for positive control

NUMBER OF CELLS EVALUATED: 1.6, 1.6 and 20000 cells per well plated for survival, viability and 6TG resistance respectively.

DETERMINATION OF CYTOTOXICITY
- Method: Percentage Relative Survival
Cloning efficiency (CE) = P / No of cells plated per well; and as an average of 1.6 cells/well were plated on all survival and viability plates, CE = P/1.6.
Percentage relative survival (% RS) = [CE (test)/CE (control)] x 100.
Adjusted % RS = % RS x (Post-treatment cell concentration for test article treatment / Post-treatment cell concentration for vehicle control)

- OTHER:
Mutant Frequency (MF) per 10^6 viable cells for each set of plates was calculated as: MF = [CE (mutant)/CE (viable)] x 10^6.
Evaluation criteria:
For valid data, the test article was considered to be mutagenic in this assay if:
1. The MF at one or more concentrations was significantly greater than that of the negative control (p≤0.05)
2. There was a significant concentration-relationship as indicated by the linear trend analysis (p≤0.05)
3. If both of the above criteria were fulfilled, the results should exceed the upper limit of the last 20 studies in the historical negative control database (mean MF +/ 2 standard deviations.
Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis.
Statistics:
Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines (Robinson et al., 1990). The control log mutant frequency (LMF) was compared with the LMF from each treatment concentration and the data were checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: No marked changes in osmolality or pH were observed in the Range-Finder at the highest concentration tested (1250 μg/mL), compared to the concurrent vehicle controls.
- Precipitation: Yes
- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES:
In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9 ranging from 39.06 to 1250 μg/mL (a precipitating concentration based on data previously generated in a solubility assessment). Upon addition of the test item to the cultures and following the 3 hour treatment incubation period, precipitate was observed at 1250 μg/mL in the absence and presence of S-9. The highest concentrations to give >10% relative survival (RS) were 78.13 μg/mL in the absence of S-9 and 312.5 μg/mL in the presence of S-9, which gave 60% and 96% RS, respectively

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g
. 95%)
The historical control ranges for the last 20 experiments performed in this laboratory are as follows:
- Negative (solvent/vehicle) historical control data:
Vehicle Controls
In the absence of S-9
Mean: 4.52 mutants per 10^6 viable cells, Range* = 0.80 to 8.24 mutants per 10^6 viable cells.
In the presence of S-9
Mean: 5.26 mutants per 10^6 viable cells, Range* = 1.30 to 9.22 mutants per 10^6 viable cells.
*Range = Mean ± 2 x SD.
- Positive historical control data:
NQO 0.15 μg/mL in the absence of S-9:
Mean: 43.04 mutants per 10^6 viable cells, Range* = 1.08 to 84.99 mutants per 10^6 viable cells.
NQO 0.2 μg/mL in the absence of S-9:
Mean: 56.23 mutants per 10^6 viable cells, Range* = 7.85 to 104.62 mutants per 10^6 viable cells.
B[a]P 2 μg/mL in the presence of S-9:
Mean: 28.32 mutants per 10^6 viable cells, Range* = 0 to 58.85 mutants per 10^6 viable cells.
B[a]P 3 μg/mL in the presence of S-9:
Mean: 42.66 mutants per 10^6 viable cells, Range* = 8.06 to 77.25 mutants per 10^6 viable cells.
*Range = Mean ± 2 x SD.

MUTATION EXPERIMENT
- In the Mutation Experiment twelve concentrations, ranging from 40 to 200 μg/mL, in the absence of S-9 and ranging from 50 to 500 μg/mL in the presence of S-9, were tested. Upon addition of the test item to the cultures, precipitate was observed at 220 μg/mL and above in the presence of S-9. However, no post-treatment precipitation was observed in either the absence or presence of S-9. Seven days after treatment, the highest seven concentrations in the absence of S-9 (130 to 200 μg/mL) and the highest four concentrations in the presence of S-9 (300 to 500 μg/mL) were considered too toxic for selection to determine viability and 6TG resistance. In addition intermediate concentrations of 220 and 260 μg/mL in the presence of S-9 were not selected due to either excessive heterogeneity/marked toxicity. All other concentrations were selected in the absence and presence of S-9. The highest concentrations analysed were 120 μg/mL in the absence of S-9 and 280 μg/mL in the presence of S-9, which gave 10% and 13% RS, respectively.
- When tested up to toxic concentrations, no statistically significant increases in MF were observed following treatment with test item at any concentration analysed in the absence and presence of S-9 and there were no statistically significant linear trends, indicating a clear negative result.

Table 7.6.1/1: Range-finder experiment - 3 h treatment in the absence and presence of S-9

 

Concentration (μg/mL)

%RS (Percent Relative Survival)

3 hour treatment –S-9

3 hour treatment +S-9

0

100

100

39.06

94

99

78.13

60

75

156.3

3

87

312.5

0

96

625

0

0

1250 P, PP

0

0

P Precipitation noted at time of treatment

PP Precipitation noted at end of treatment incubation period

 

Table 7.6.1/2: Mutation experiment - 3 h treatment in the absence and presence of S-9

 

3 hour treatment –S-9

3 hour treatment +S-9

Concentration (μg/mL)

%RS (Percent Relative Survival)

MF §

Concentration (μg/mL)

%RS (Percent Relative Survival)

MF §

0

100

8.78

0

100

6.01

40

103

8.46 NS

50

106

6.37 NS

60

85

10.81 NS

100

104

4.90 NS

80

80

7.02 NS

150

92

5.67 NS

100

78

3.26 NS

200

70

8.09 NS

120

10

4.38 NS

240 P

25

6.81 NS

 

 

 

280 P

13

9.03 NS

NQO 0.15

37

56.79

B[a]P 2

30

61.14

NQO 0.2

36

50.38

B[a]P 3

5

71.35

Linear trend: Not Significant (negative trend) – 3 hour absence of S-9

Linear trend: Not Significant – 3 hour presence of S-9

Conclusions:
Under the test conditions, test item did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to the limit of toxicity, in the absence and presence of a rat liver metabolic activation system.
Executive summary:

In an in vitro mammalian cell gene mutation test performed according to OECD Guideline 476 and in compliance with GLP, L5178Y tk+/-(3.7.2C) mouse lymphoma cells were exposed to test item for 3 h at the following concentrations:

Range-Finder Experiment: 39.06, 78.13, 156.3, 312.5, 625 and 1250 μg/mL, with and without S9 mix

Mutation Experiment:

Without S9: 40, 60, 80, 100, 120, 130, 140, 150, 160, 170, 180 and 200 μg/mL

With S9: 50, 100, 150, 200, 220, 240, 260, 280, 300, 350, 400 and 500 μg/mL

Vehicle and positive control groups were also included in each mutagenicity test. Metabolic activation system used in this test was 2 % S9 mix (final concentration). S9 fraction was prepared from liver homogenates of rats treated with Aroclor 1254.

 

In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9 ranging from 39.06 to 1250 μg/mL (a precipitating concentration based on data previously generated in a solubility assessment). Post-treatment precipitation was observed at 1250 μg/mL in the absence and presence of S-9. The highest concentrations to give>10% relative survival (RS) were 78.13 μg/mL in the absence of S-9 and 312.5 μg/mL in the presence of S-9, which gave 60% and 96% relative survival (RS), respectively.

 

In the Mutation Experiment twelve concentrations, ranging from 40 to 200 μg/mL, in the absence of S-9 and ranging from 50 to 500 μg/mL in the presence of S-9, were tested. No post-treatment precipitation was observed. Seven days after treatment the highest concentrations analysed to determine viability and 6TG resistance were 120 μg/mL in the absence of S-9 and 280 μg/mL in the presence of S-9, which gave 10% and 13% RS, respectively.

 

Vehicle and positive control treatments were included in the Mutation Experiment in the absence and presence of S-9. Mean mutant frequencies (MF) in vehicle control cultures fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (NQO) (without S-9) and benzo(a)pyrene (B[a]P) (with S-9). Therefore the study was accepted as valid.

 

When tested up to toxic concentrations, no statistically significant increases in MF were observed following treatment with test item at any concentration analysed in the absence and presence of S-9 and there were no statistically significant linear trends, indicating a clear negative result.

 

Under the test conditions, test item did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to the limit of toxicity, in the absence and presence of a rat liver metabolic activation system.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
30 September to 12 November 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD test guideline No. 487. Read-across substance
Justification for type of information:
See read-across justification in section 13
Reason / purpose:
read-across source
Reason / purpose:
read-across: supporting information
Key result
Species / strain:
lymphocytes: human peripheral blood lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Solubility: DMSO was used as the vehicle based on the solubility of the test substance and compatibility with the target cells. In a solubility test conducted at BioReliance, the test substance was soluble in DMSO at a concentration of approximately 500 mg/mL, the maximum concentration tested for solubility.
- Effects of pH: The pH of the highest concentration of test substance in treatment medium was 7.5.
- Effects of osmolality: The osmolality in treatment medium of the highest dose level tested, 5000 μg/mL, was 345 mmol/kg. The osmolality in treatment medium of the lowest precipitating dose level, 500 μg/mL, was 427 mmol/kg. The osmolality in treatment medium of the highest soluble dose level, 150 μg/mL, was 428 mmol/kg. The osmolality of the vehicle (DMSO) in the treatment medium was 434 mmol/kg. The osmolality of the test substance dose levels in treatment medium is acceptable because it did not exceed the osmolality of the vehicle by more than 20%.
- Precipitation: Yes

PRELIMINARY TOXICITY TEST
The test substance was soluble in DMSO at all concentrations tested. Visible precipitate was observed in treatment medium at dose levels ≥ 500 μg/mL, while dose levels ≤ 150 μg/mL were soluble in treatment medium at the beginning of the treatment period. At the conclusion of the treatment period, visible precipitate was observed in treatment medium at dose levels ≥ 1500 μg/mL, while dose levels ≤ 500 μg/mL were soluble in treatment medium in all treatment conditions. Also at the conclusion of the treatment period, hemolysis was observed at dose levels ≥ 500 μg/mL in the non-activated 4-hour exposure group, and at dose levels ≥ 1500 μg/mL in the S9-activated 4-hour and the non-activated 24-hour exposure groups.
Substantial cytotoxicity [≥ 50% cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control] was observed at dose levels ≥ 150 μg/mL in the non-activated 4 and 24-hour exposure groups, and at dose levels ≥ 500 μg/mL in the S9-activated 4-hour exposure group.

MAIN TEST
In the micronucleus assay, the test substance was soluble in DMSO at all concentrations tested. Visible precipitate was observed in treatment medium at dose levels ≥ 225 μg/mL, while dose levels ≤ 150 μg/mL were soluble in treatment medium at the beginning of the treatment period. At the conclusion of the treatment period, in the S9-activated 4-hour exposure group, visible precipitate was observed in treatment medium at dose levels ≥ 225 μg/mL, while dose levels ≤ 150 μg/mL were soluble in treatment medium. In the non-activated 4 and 24-hour exposure groups, all dose levels were soluble in the treatment medium at the conclusion of the treatment period. The pH of the highest concentration of test substance in treatment medium was 7.5.
4 hours exposure with 20 hours recovery time, without metabolic activation: The dose levels selected for analysis of micronucleus were 50, 100, and 150 μg/mL. At the highest test concentration, 150 μg/mL, cytotoxicity was 53% relative to the vehicle control. The percentage of cells with micronuclei in the test substance-treated group was not significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher's Exact test). The percentage of micronucleated cells in the MMC (positive control) group (3.2%) was statistically significant (p ≤ 0.01, Fisher's Exact test).
4 hours exposure with 20 hours recovery time, with metabolic activation: The dose levels selected for analysis of micronucleus were 50, 150, and 225 μg/mL. At the highest test concentration, 225 μg/mL, cytotoxicity was 5% relative to the vehicle control. The percentage of cells with micronuclei in the test substance-treated group was not significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher's Exact test). The percentage of micronucleated cells in the CP (positive control) group (1.3%) was statistically significant (p ≤ 0.01, Fisher's Exact test).
24 hours exposure without recovery, without metabolic activation: The dose levels selected for analysis of micronucleus were 25, 50, and 70 μg/mL. At the highest test concentration, 70 μg/mL, cytotoxicity was 58% relative to the vehicle control. The percentage of cells with micronuclei in the test substance-treated group was not significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher's Exact test). The percentage of micronucleated cells in the VB (positive control) group (2.2%) was statistically significant (p ≤ 0.01, Fisher's Exact test).

HISTORICAL CONTROL DATA (with ranges, means and standard deviation
- Positive historical control data: Mitomycin C (range: 1.2-9.0%, mean: 4.120 ± 1.797%); Cyclophosphamide (range: 0.8-2.9%, mean: 1.496 ± 0.420%)
- Negative (solvent/vehicle) historical control data: without metabolic activation (range: 0.1-1.6%, mean: 0.364 ± 0.254%); with metabolic activation (range: 0.0-1.5%, mean: 0.345 ± 0.249%)

None

Conclusions:
Under the test conditions, Lavender Oil was concluded to be negative for the induction of micronuclei in the non-activated and S9-activated test systems in the in vitro mammalian micronucleus test using human peripheral blood lymphocytes. Therefore, the registered substance Lavandin oil is not considered as clastogenicin mammalian cells.
Executive summary:

In an in vitro micronucleus test performed according to OECD Guideline 487 and in compliance with GLP, cultured Human peripheral blood lymphocytes were exposed to test substance in the presence and absence of a metabolic activation system.

 

In the preliminary toxicity and the micronucleus assays, HPBL cells were treated for 4 and 24 hours in the non-activated test system and for 4 hours in the S9-activated test system. All cells were harvested 24 hours after treatment initiation.

 

Preliminary Toxicity Test

4 hours exposure with 20 hours recovery time: 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/mL, with and without metabolic activation

24 hours exposure without recovery: 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/mL, with and without metabolic activation

 

Main test:

4 hours exposure with 20 hours recovery time: 10, 25, 50, 60, 70, 85, 100, 125 and 150 μg/mL, without metabolic activation

4 hours exposure with 20 hours recovery time: 50, 100, 150, 225, 250, 275, 300, 325, 350, 375, 400 and 450 μg/mL, with metabolic activation

24 hours exposure without recovery: 10, 25, 50, 60, 70, 85, 100 and 125 μg/mL, without metabolic activation

 

Dimethyl sulfoxide (DMSO) was used as the vehicle based on the solubility of the test substance and compatibility with the target cells. In a solubility test, the test substance was soluble in DMSO at a concentration of approximately 500 mg/mL, the maximum concentration tested for solubility.

 

In the preliminary toxicity assay, substantial cytotoxicity [≥ 50% cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control] was observed at dose levels ≥ 150 μg/mL in the non-activated 4 and 24-hour exposure groups, and at dose levels ≥ 500 μg/mL in the S9-activated 4-hour exposure group. Based on these findings, the doses chosen for the micronucleus assay ranged from 10 to 150 μg/mL for the non-activated 4-hour exposure group, from 50 to 450 μg/mL for the S9-activated 4-hour exposure group, and from 10 to 125 μg/mL for the non-activated 24-hour exposure group.

 

In the micronucleus assay, substantial cytotoxicity was observed at 150 μg/mL in the non-activated 4-hour exposure group, at dose levels ≥ 275 μg/mL in the S9-activated 4-hour exposure group, and at dose levels ≥ 70 μg/mL in the non-activated 24-hour exposure group. The highest dose analyzed under each treatment condition either exceeded the limit of solubility in treatment medium at the conclusion of the treatment period or produced 50 to 60% reduction in CBPI, which met the dose limit as recommended by testing guidelines for this assay. A minimum of 1000 binucleated cells from each culture were examined and scored for the presence of micronuclei.

 

The percentage of cells with micronucleated binucleated cells in the test substance-treated groups was not statistically significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher’s Exact test). The results for the positive and negative controls indicate that all criteria for a valid assay were met.

Under the test conditions, Lavender Oil was concluded to be negative for the induction of micronuclei in the non-activated and S9-activated test systems in the in vitro mammalian micronucleus test using human peripheral blood lymphocytes. Therefore, the registered substance Lavandin oil is not considered as clastogenic in mammalian cells.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Table 7.6/1: Summary of genotoxicity tests

 

Test n°

Test / Guideline

Reliability

Focus

Strains tested

Metabolic activation

Test concentration

Statement

1

Ames Test

(OECD 471)

K, rel. 1, 2019

Gene mutation

TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

-S9

+S9

Up to cytotoxic concentrations

-S9 : non mutagenic

+S9 : non mutagenic

2

Ames Test

(OECD 471)

SS, rel. 1, 2014

Gene mutation

 E. Coli

TA 1535,

TA 1537,

TA 98,

TA 100

-S9

+S9

 

Up to cytotoxic concentrations

 -S9 : non mutagenic

+S9 : non mutagenic

 3

MNT (OECD 487)

K, rel. 1, 2015

 Chromosome aberration

  Human Lymphocytes

-S9

+S9

  Up to cytotoxic concentrations

-S9 : non clastogenic

+S9 : non clastogenic

 4

 

L5178Y cells

/HPRT test (OECD 476)

K, rel.1, 2017

 Mammalian gene mutation

 Mouse Lymphoma cells

-S9

+S9

 Up to cytotoxic concentration

-S9 : non mutagenic

+S9 : non mutagenic

 

Gene mutation Assays (Tests n° 1, 2 and 4):

- A Bacterial Reverse mutation Assay (Ames test n°1) was performed according to OECD test guideline No 471 with the test substance (See Table 1). No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose, either in the presence or absence of metabolic activation. The substance does not induce gene mutations in bacteria under the test condition whereas all positive control chemicals (with and without metabolic activation) induced significant increase of colonies. The test substance is therefore considered as non-mutagenic according to the Ames test. These results are supported by an Ames test (n°2) performed according to OECD test guideline No 471 with the source substance (See Table 1). No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose, either in the presence or absence of metabolic activation. The source substance does not induce gene mutations in bacteria under the test conditions. Therefore, both thesource and the target substances are considered as non-mutagenic according to the Ames test.

- Inability to produce gene mutation was confirmed in mammal cells using an in vitro gene mutation assay in L5178Y tk+/-(3.7.2C) mouse lymphoma cells (L5178Y TK+/- /MLA test) (Test n°4). None of the dose levels up to the cytotoxicity limit with the test substance, either in the presence or absence of metabolic activation, induced significant mutant frequency increases in the initial or repeat experiments whereas both positive control chemicals (with and without metabolic activation) induced significant mutant frequency increases. Therefore, the test substance is considered as negative for inducing gene mutations at the hprt locus in L5178Y mouse lymphoma cells when tested up to the limit of toxicity. This result confirms the results of the Ames test and extends the non-mutagenic effect of the test substance to mammalian cells.

Chromosomal aberration (Test n°3)

The clastogenic potential of the test material was determined with the source substance (test n°3) using an in vitro chromosome aberration test in Human lymphocytes, which measures the potential of a substance to increase the incidence of structural chromosome aberrations in cultured Human lymphocytes.

None of the dose levels up to the cytotoxicity limit with the supporting substance, either with or without metabolic activation, induced significant increases in the frequency of cells with aberrations in either of two experiments. The source substance does not induce structural aberrations in the chromosomes of Human lymphocytes under activation and non-activation conditions, whereas both positive control chemicals (with and without metabolic activation) induced significant increases in the frequency of aberrant cells. Therefore, both the source and the target substancesare considered as negative for inducing chromosomal aberration in Human lymphocytes in vitro under activation and non-activation conditions used in this assay.

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification for human health according to the Regulation (EC) No. 1272/2008 (CLP).

 

Self classification:

Based on the available data, no additional classification is proposed regarding genetic toxicity according to the CLP and to the GHS.